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目的探讨雌、孕激素激活磷酸化信号传导通路后,对卵巢上皮性癌(卵巢癌)细胞转移抑制基因nm23-H1蛋白表达的影响。方法采用不同浓度的17β雌二醇(雌激素组)及醋酸甲羟孕酮(孕激素组)分别作用于卵巢透明细胞癌细胞株ES-2细胞,以二甲基亚砜作为对照(对照组)。采用划痕实验观察各组细胞迁移能力的变化;采用穿膜小室(Transwell小室)体外侵袭实验观察各组细胞侵袭能力的变化;采用蛋白印迹法检测各组细胞nm23-H1蛋白表达及蛋白激酶B(AKT)蛋白、磷酸化蛋白激酶B(pAKT)蛋白表达的变化及其与时间、剂量的相关性;采用RNA干扰技术观察转染AKT小分子干扰RNA(siRNA)质粒后,各组细胞nm23-H1蛋白表达的变化及其侵袭能力的变化。结果划痕实验24h时,各组ES-2细胞均向划痕处弥合,划痕间距雌激素组为(1·39±0·08)mm,孕激素组为(1·96±0·07)mm,对照组为(1·65±0·12)mm;雌激素组明显小于对照组(P=0·029),而孕激素组明显大于对照组(P=0·014)。Transwell小室培养12h时,穿过微孔膜的细胞雌激素组为(119±13)个,孕激素组为(78±8)个,对照组为(92±16)个;雌激素组明显多于对照组(P=0·015),而孕激素组明显少于对照组(P=0·006)。蛋白印迹法检测结果显示,17β雌二醇降低ES-2细胞nm23-H1蛋白表达的作用与剂量和时间呈明显负相关关系(P=0·020,P=0·001),醋酸甲羟孕酮增加ES-2细胞nm23-H1蛋白表达的作用与剂量和时间呈明显正相关关系(P=0·003,P=0·002);17β雌二醇增加ES-2细胞pAKT蛋白表达的作用与剂量和时间呈明显正相关关系(P=0·001,P=0·007),醋酸甲羟孕酮降低ES-2细胞pAKT蛋白表达的作用与剂量和时间呈明显负相关关系(P=0·012,P=0·039);而17β雌二醇和醋酸甲羟孕酮对ES-2细胞AKT蛋白的表达均无明显影响(P>0·05)。AKTsiRNA干扰后,减弱了17β雌二醇对ES-2细胞nm23-H1蛋白表达的降调节作用(P=0·076)和促进ES-2细胞穿过Transwell小室的能力(P=0·208),同样也减弱了醋酸甲羟孕酮对ES-2细胞nm23-H1蛋白表达的升调节作用(P=0·105)和抑制ES-2细胞穿过Transwell小室的能力(P=0·497),但差异均无统计学意义。结论雌激素可通过激活磷酸化信号传导通路下调卵巢癌细胞nm23-H1蛋白的表达,参与卵巢癌的转移;而孕激素则可增加卵巢癌细胞nm23-H1蛋白的表达,抑制卵巢癌的转移。
Objective To investigate the effect of estrogen and progesterone on the expression of nm23-H1 protein in epithelial ovarian cancer (ovarian cancer) cells after activation of phosphorylation signal transduction pathway. Methods Different concentrations of 17β estradiol (estrogen group) and medroxyprogesterone acetate (progesterone acetate group) were applied to ES-2 cells of ovarian clear cell line with dimethylsulfoxide (control group ). The migration of cells in each group was observed by scratch test. The invasion ability of cells in each group was observed by in vitro invasion assay. The expressions of nm23-H1 protein and protein kinase B (AKT) protein and phosphorylated protein kinase B (pAKT) protein and their correlation with time and dose. After transfection with AKT siRNA, the expression of nm23- H1 protein expression and invasion ability changes. Results ESR cells of ES-2 cells were all scratched at scratches 24h after scratch test. The interval between the two groups was (1.39 ± 0.08) mm in estrogen group and (1.96 ± 0.07) in progesterone group ) mm, and the control group was (1.65 ± 0.12) mm. The estrogen group was significantly smaller than the control group (P = 0.0299), while the progesterone group was significantly larger than the control group (P = 0.014). Transwell chamber cultured 12h, the cell membrane estrogen group (119 ± 13), progesterone group (78 ± 8), the control group (92 ± 16); estrogen group was significantly more In the control group (P = 0.015), while the progestin group was significantly less than the control group (P = 0.006). The results of Western blotting showed that the effect of 17β-estradiol on the expression of nm23-H1 in ES-2 cells was negatively correlated with the dose and time (P = 0.020, P = 0.001) The effect of ketone on the expression of nm23-H1 in ES-2 cells was positively correlated with the dose and time (P = 0.003, P = 0.002). The effect of 17β-estradiol on pAKT protein expression in ES-2 cells (P = 0.001, P = 0.007). Medroxyprogesterone acetate reduced the expression of pAKT protein in ES-2 cells in a dose-and-time-dependent manner (P = 0 · 012, P = 0 · 039). However, 17β estradiol and medroxyprogesterone acetate had no significant effect on AKT protein expression in ES-2 cells (P> 0.05). AKT siRNA reduced the down-regulation of nm23-H1 expression in ES-2 cells (P = 0.076) and the ability of ES-2 cells to pass through Transwell chambers (P = 0.208) Also attenuated the upregulation of nm23-H1 protein expression in ES-2 cells (P = 0.05) and the ability of ES cells to pass through Transwell chambers (P = 0.497) , But the differences were not statistically significant. CONCLUSIONS: Estrogen can down-regulate the expression of nm23-H1 protein in ovarian cancer cells by activating the phosphorylation signal transduction pathway and participate in the metastasis of ovarian cancer. However, progestin can increase the expression of nm23-H1 protein in ovarian cancer cells and inhibit the metastasis of ovarian cancer.